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Nitric Oxide-Dependent Ribosomal RNA
Cleavage Is Associated with Inhibition of
Ribosomal Peptidyl Transferase Activity in
ANA-1 Murine Macrophages
Charles Q. Cai, Hongtao Guo, Rebecca A. Schroeder, Cecile
Punzalan and Paul C. Kuo
J Immunol 2000; 165:3978-3984; ;
doi: 10.4049/jimmunol.165.7.3978
http://www.jimmunol.org/content/165/7/3978
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Copyright © 2000 by The American Association of
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References
Nitric Oxide-Dependent Ribosomal RNA Cleavage Is
Associated with Inhibition of Ribosomal Peptidyl Transferase
Activity in ANA-1 Murine Macrophages1
Charles Q. Cai,* Hongtao Guo,* Rebecca A. Schroeder,† Cecile Punzalan,* Paul C. Kuo2*
E
xpression of inducible NO synthase (iNOS)3 protein in
settings rich in pro-inflammatory cytokines or endotoxin
is associated with multiple alterations in cellular function.
These include inhibition of protein synthesis, induction of cytostasis, modification of transcriptional programs, inhibition of mitochondrial respiration, and altered cellular redox state (1). In some
instances, the underlying mechanism is related to NO-mediated
S-nitrosation of key protein thiol groups with subsequent alteration
in enzymatic function or protein binding properties (2, 3). Alternatively, NO alters the cellular redox balance by virtue of its oxidative properties, and induces downstream counterregulatory
events (4, 5). However, the association between high concentrations of NO, as expressed by iNOS, and inhibition of total protein
synthesis has not been extensively examined. The effect of NO
upon global cellular translation, gene transcription, or cellular architecture critical to the protein synthetic process is unknown. In
particular, the effect of NO upon the ribosomal scaffolding, which
is critical to initiation and potentiation of protein translation, has
not been examined.
In this study, using a model of endotoxin-mediated iNOS expression in ANA-1 murine macrophages, we demonstrate that NO
inhibits total protein synthesis and cell proliferation without alter-
*Departments of Surgery and †Anesthesiology, Georgetown University Medical Center, Washington, D.C. 20007
Received for publication February 8, 2000. Accepted for publication July 14, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Clowes Faculty Development Award from the
American College of Surgeons (to P.C.K.) and National Institutes of Health Grant
AI44629 (to P.C.K.).
2
Address correspondence and reprint requests to Dr. Paul C. Kuo, Georgetown University Medical Center, Department of Surgery, 4 PHC, 3800 Reservoir Rd, N.W.,
Washington, D.C. 20007. E-mail address: [email protected]
3
Abbreviations used in this paper: iNOS, inducible NO synthase; SNAP, S-nitrosoN-acetylpenicillamine; L-NAME, N G-nitro-L-arginine methyl ester.
Copyright © 2000 by The American Association of Immunologists
ation in global gene transcription. However, induction of iNOS
and inhibition of total protein synthesis in this cell line are associated with a specific and reproducible cleavage pattern in 28S and
18S rRNA that is both NO- and time-dependent. We examined
enzymatic function of the 60S ribosome, which is largely composed of intact 28S and 18S rRNA. Levels of 60S ribosome and
60S ribosome-associated peptidyl transferase activity, an essential
part of peptide chain elongation are both significantly depressed in
the settings of both LPS-mediated endogenous NO synthesis and
exposure to an exogenous NO donor, S-nitroso-N-acetylpenicillamine (SNAP). We conclude that NO-mediated inhibition of total
protein synthesis is the result of decreased 60S ribosome-associated peptidyl transferase activity.
Materials and Methods
Cell culture
ANA-1 murine macrophages, a gift from Dr. George Cox (Uniformed Services University of the Health Sciences, Bethesda, MD), were maintained
in DMEM with 5% heat-inactivated FCS. LPS (Escherichia coli serotype
0111:B4; 0 –10,000 ng/ml) was added to the medium to induce NO synthesis. In selected instances, the competitive substrate inhibitor of NOS,
N G-nitro-L-arginine methyl ester (L-NAME; 4 mM), alone or together with
LPS and the NO donors, SNAP or S-nitroso-N-acetylcysteine were added.
Cells were harvested after incubation for 2–20 h at 37°C in 95% O2/5%
CO2. To determine cell viability, cells were washed with ice-cold PBS
three times, stained with 0.2% trypan blue solution (w/v) for 10 min, and
viable cells were counted under the light microscope. Cell viability was
routinely ⬎95% under all treatment conditions.
Determination of NO synthesis
NO release from cells in culture was quantified by measurement of the NO
metabolite, nitrite, using the technique of Snell and Snell (6). To reduce
nitrate to nitrite, 200 ␮l conditioned media was incubated in the presence
of 1.0 U nitrate reductase, 50 ␮M NADPH, and 5 ␮M flavin adenine
dinucleotide. Sulfanilamide (1%) in 0.5N hydrochloric acid (50% v/v) was
then added. After a 5-min incubation at room temperature, an equal volume
of 0.02% N-(1-(naphthyl))ethylenediamine was added; following incubation at room temperature for 10 min, absorbance at 570 nm was compared
with that of a NaNO2 standard.
0022-1767/00/$02.00
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NO can regulate specific cellular functions by altering transcriptional programs and protein reactivity. With respect to global
cellular processes, NO has also been demonstrated to inhibit total protein synthesis and cell proliferation. The underlying mechanisms are unknown. In a system of ANA-1 murine macrophages, iNOS expression and NO production were induced by exposure
to endotoxin (LPS). In selected instances, cells were exposed to an exogenous NO donor, S-nitroso-N-acetylpenicillamine or a
substrate inhibitor of NO synthesis. Cellular exposure to NO, from both endogenous and exogenous sources, was associated with
a significant time-dependent decrease in total protein synthesis and cell proliferation. Gene transcription was unaltered. In parallel
with decreased protein synthesis, cells exhibited a distinctive cleavage pattern of 28S and 18S rRNA that were the result of two
distinct cuts in both 28S and 18S rRNA. Total levels of intact 28S rRNA, 18S rRNA, and the composite 60S ribosome were
significantly decreased in the setting of cell exposure to NO. Finally, 60S ribosome-associated peptidyl transferase activity, a key
enzyme for peptide chain elongation, was also significantly decreased. Our data suggest that NO-mediated cleavage of 28S
and 18S rRNA results in decreased 60S ribosome associated peptidyl transferase activity and inhibition of total protein
synthesis. The Journal of Immunology, 2000, 165: 3978 –3984.
The Journal of Immunology
RNA and DNA extraction and Northern blot analysis
Total RNA was purified from cultured ANA-1 macrophages with the use
of TRIzol Reagent (Life Technologies, Rockville, MD). The purified total
RNA (5 ␮g) was subjected to electrophoresis on a 1% (w/v) agarose gel for
3 h at 80 v. The RNA was blotted onto Nytran nylon transfer membrane
(Schleicher & Schuell, Keene, NH) with 10⫻ SSC (1.5 M NaCl/0.15 M
sodium citrate) prepared with diethyl pyrocarbonate-treated water, then
cross-linked to the membrane by UV radiation. cDNA probes complementary to murine 28S and 18S rRNA were prepared by PCR amplification
with primers based on their cDNA sequence and randomly labeled with
[32P]dCTP. Hybridization of the labeled probe was performed in hybridization solution (50% formatted, 5⫻ SSC, 5 mM EDTA, 20 mM sodium
phosphate buffer (pH 7), 1% SDS, 200 ␮g/ml salmon sperm DNA, and 5⫻
Denhardt’s solution) at 42°C overnight with 3 h of prehybridization at
42°C. The blots were washed briefly with 2⫻ SSC containing 0.1% SDS
at room temperature, once for 15 min with 0.5⫻ SSC containing 0.1% SDS
at 42°C, and once for 15 min with 0.2⫻ SSC containing 0.1% SDS at 50°C
and 65°C. Autoradiographs were exposed at room temperature. Genomic
DNA was purified from cultured ANA-1 macrophages with the DNAzol
Reagent (Life Technologies). The purified genomic DNA were subjected to
electrophoresis on a 0.6% (w/v) agarose gel for 3 h at 100 V.
Determination of protein synthesis
Cell proliferation assays
ANA-1 macrophages were labeled with 5 ␮Ci/ml [3H]thymidine for 20 h
in the presence or absence of LPS. The plates were treated with 0.5 ml/well
ice-cold 10% trichloroacetic acid and kept at 4°C overnight. The plates
were washed three times with distilled water and dried at room temperature. Cells were collected with 0.5 ml/well 0.33 mM NaOH and 300 ␮1
solution was transferred into a scintillation vial containing scintillation
fluid. [3H]Thymidine incorporation into DNA was measured using a scintillation counter.
crocentrifuge. The supernatant formed the total ribosomal fraction and a
portion was used for peptidyl transferase activity. The remaining supernatant was layered onto a linear 10 – 45% sucrose gradient containing 25 mM
Tris-HCl, 80 mM KCl, 4 mM MgCl2, and 20 mM sodium fluoride. Following centrifugation for 4 h in a Beckman (Fullerton, CA) SW-41 rotor,
ribosomal profiles were monitored by continuously measuring A280.
Ribosome-associated peptidyl transferase activity
Peptidyl transferase activity was measured in ANA-1 macrophage total
ribosomal fractions using a peptidyl transferase reaction with full-length
formyl-[3H]Met RNA as a donor substrate (7). Typically, cell fractions
were incubated with 2.5 pmol formyl-[3H]Met RNA in 40 ␮l buffer containing 20 mM Tris-HCl (pH 8.0), 400 mM KCl, 20 mM MgCl2, and 1 mM
puromycin. Reactions were initiated by adding 20 ␮l cold methanol and
incubated for 20 min at 0°C. To terminate the reaction, 10 ␮l 4 M KOH
was added and incubated for an additional 20 min at 37°C. After the addition of 200 ␮l 1 M KH2PO4, the reaction mixture was extracted with 1
ml ethyl acetate. A portion of the extract was mixed with scintillation
mixture and counted. Peptidyl transferase activity is expressed as fmol/mg
protein/min.
Statistical analysis
Data are presented as mean ⫾ SEM of three or four experiments each
performed in triplicate. Statistical analysis was performed using the Student t test or rank sum test, as appropriate.
Results
LPS and NO synthesis in ANA-1 macrophages
Following stimulation with 10 –10,000 ng/ml LPS for a period of
20 h, ANA-1 murine macrophage production of NO was determined by measuring levels of nitrite and nitrate in the culture media (Fig. 1). The dose response was plotted in a semilogarithmic
fashion. In the absence of LPS, the nitrite level in the media was
9.9 ⫾ 1.0 nmol/mg protein. NO production increased in a significant dose-related fashion at LPS concentrations of 10, 100, 1,000,
and 10,000 ng/ml ( p ⬍ 0.01 vs control for LPS ⫽ 100, 1,000, and
10,000 ng/ml). The competitive substrate inhibitor L-NAME (4 mM)
was added. In the presence of L-NAME alone, nitrite production was
not significantly altered from that of control cells (10.1 ⫾ 1.1 nmol/
Nuclear run-on analysis
A total of 100 ␮l ANA-1 macrophage nuclei was incubated for 5 min at
30°C with 150 ␮Ci of [32P]rUTP (800 Ci/mmol) in 100 ␮l 10 mM Tris HCl
(pH 8.0), 5 mM MgCl2, 300 mM KCl, and 5.0 mM (each) ATP, CTP, and
GTP. Labeled RNA was isolated by the acid-guanidinium thiocyanate
method. Before ethanol precipitation, labeled RNA was treated with 0.2 M
NaOH for 10 min on ice. The solution was neutralized by the addition of
HEPES (acid free) to a final concentration of 0.24 M. After ethanol precipitation, the RNA pellet was re-suspended in 10 mM N-tris(hydroxymethyl)methyl-2 minoethanesulfonic acid (pH 7.4), 0.2% SDS, and 10 mM
EDTA. Target DNA for cyclophillin, ␤-actin, and GAPDH was spotted
onto nylon membranes with a slot blot apparatus. ␭-Bacteriophage DNA
served as negative controls. Hybridization was performed at 42°C for 48 h
with 5 ⫻ 106 cpm of labeled RNA in hybridization buffer (50% formamide,
4⫻ SSC, 0.1% SDS, 5⫻ Denhardt’s solution, 0.1 M sodium phosphate (pH
7.2), and 10 ␮g/ml salmon sperm DNA). After hybridization, the membranes were washed twice at room temperature in 2⫻ SSC and 0.1% SDS,
and three times at 56°C in 0.1⫻ SSC and 0.1% SDS. The membranes were
then exposed to x-ray film and scanned.
Cellular ribosome profile
ANA-1 murine macrophages were isolated in ice-cold PBS. The cells were
pelleted at maximum speed in a microcentrifuge and resuspended in hypotonic buffer (40 mM Tris-HCl (pH 7.4), 20 mM KCl, 3 mM MgCl2, 20
mM sodium fluoride, and 150 mM sucrose) and kept on ice for an additional 10 min. Following cell lysis in 1% Triton X-100 and 1% deoxycholate, the preparation was vortexed and centrifuged for 1 min in a mi-
FIGURE 1. NO dose-response relationship in LPS-treated ANA-1 murine macrophages. Murine macrophages were exposed to 1–10,000 ng/ml
LPS for a period of 20 h. In selected instances, 4 mM L-NAME was also
added as a competitive substrate inhibitor of NO synthesis. Results represent data from four separate experiments. ⴱ, p ⬍ 0.01; LPS vs LPS ⫹
L-NAME. ANOVA for LPS curve, p ⫽ 0.0005.
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Cells were washed with methionine-free DMEM three times and incubated
for 20 min in short-term medium (methionine-free DMEM containing 5%
dialyzed FCS) in a humidified 37°C, 5% CO2 incubator to deplete intracellular pools of methionine. Medium was then replaced with fresh shortterm labeling medium containing 10 ␮Ci/ml [35S]methionine. After 15-,
30-, 60-, 120-, and 240-min labeling, cells were washed with PBS twice
and lysed in TE buffer supplied with 1% SDS. The cell lysis was then
passed through a 21 gauge needle several times. A total of 50 ␮l labeled
cell lysis was added to 0.5 ml BSA (0.1 mg/ml) on ice and an equal amount
(0.5 ml) of ice-cold 20% trichloroacetic acid was added. The solution was
vortexed vigorously and incubated for 30 min on ice. The precipitate was
collected on filter papers and was washed once with ice-cold 10% TCA and
twice with 95–100% ethanol. When dry, the filter papers were transferred
to a scintillation vial containing 4 ml scintillation fluid. Radioactivity was
then measured.
3979
3980
NO AND RIBOSOMAL RNA DEGRADATION
mg). In cells treated with LPS ⫹ L-NAME, NO production was ablated at all concentrations of LPS (10 –10,000 ng/ml) and did not
significantly differ from that of control cells. In addition, trypan blue
exclusion and media levels of lactate dehydrogenase were determined
as measures of cell viability. Under all treatment conditions, cell viability was routinely ⬎95%, and LDH levels did not differ among the
various treatment groups. These data indicate that LPS-mediated NO
production is dose-dependent and does not alter cell viability. Subsequent studies use 100 ng/ml LPS and 4 mM L-NAME, unless stated
otherwise.
Cell proliferation and total protein synthesis
FIGURE 2. A, Total cellular protein synthesis in ANA-1 murine macrophages. ANA-1 murine macrophages were exposed to 100 ng/ml LPS for
a period of 20 h and pulsed with [35S]methionine. Radioactivity was determined following the designated time period; measured values were cor-
rected for total cell protein. In selected instances, 4 mM L-NAME or 400
␮M SNAP were added. Results represent data from four separate experiments. ⴱ, p ⬍ 0.05; LPS vs control and LPS ⫹ L-NAME. #, p ⬍ 0.05;
SNAP vs LPS, control, and LPS ⫹ L-NAME. B, Total cellular protein
synthesis and NO production in ANA-1 murine macrophages. ANA-1 murine macrophages were exposed to 100 ng/ml LPS for a designated period
of incubation and pulsed with [35S]methionine. Radioactivity was determined following 2 h and corrected for total cell protein. Nitrite production
was simultaneously determined in LPS-treated cells. Results represent data
from three separate experiments ( p ⬍ 0.05, LPS vs control and LPS ⫹
L-NAME). C, Total cellular protein synthesis and NO production in 100
ng/ml LPS-treated ANA-1 murine macrophages. First order linear regression analysis was performed (r 2 ⫽ 0.70).
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Following a 20-h incubation, total protein synthesis was determined by [35S]methionine incorporation in control, LPS-, LPS ⫹
L-NAME-, and SNAP 400 ␮M-treated macrophages (Fig. 2A). In
all instances, there was a significant linear, time-dependent increase in protein synthesis. In LPS-treated cells, total protein synthesis was consistently decreased by 40 –70% at all time points
when compared with those of control and LPS ⫹ L-NAME ( p ⬍
0.05 vs control and LPS ⫹ L-NAME). The greatest incremental
difference in protein synthesis was present following a 15-min
pulse. Protein synthesis in control and LPS ⫹ L-NAME cells was
not significantly different. In SNAP-treated cells, total protein synthesis was significantly decreased at all time points, in comparison
to control and LPS ⫹ L-NAME and also, when compared with LPS
alone ( p ⬍ 0.05 vs control, LPS ⫹ L-NAME, and LPS). When
cells were incubated in the presence of LPS ⫹ L-NAME ⫹ SNAP,
the protein synthesis curve was not statistically different from that of
SNAP alone (data not shown). These data suggest that NO, whether
from an exogenous or endogenous source, is associated with significant depression of total protein synthesis in ANA-1 murine
macrophages.
The assay was then repeated by determination of [35S]methionine incorporation following a 240-min pulse while varying the
duration of LPS stimulation (100 ng/ml), 0, 8, 12, 16, and 20 h
(Fig. 2B). There was no difference in protein synthesis following
8 h of LPS stimulation. However, at LPS incubation times greater
than 12 h, protein synthesis progressively decreases in a significant
fashion ( p ⬍ 0.05 vs control and LPS ⫹ L-NAME for 16 and
20 h). The time dependence of total NO synthesis was also determined and plotted in Fig. 2B. NO production increases dramatically following 12 h of LPS stimulation and reaches a near
maxium after 20 h of LPS stimulation. The time course of this
increase in NO is associated with a parallel decline in total protein
synthesis. When [35S]methionine incorporation is plotted vs nitrite
from LPS stimulated cells, an inverse linear relationship (r 2 ⫽
0.70) is present (Fig. 2C). This suggests that NO production is
inversely associated with decreased protein synthesis in LPS stimulated macrophages.
The Journal of Immunology
3981
ANA-1 cell proliferation in the setting of LPS-induced NO synthesis was determined by the incorporation of [3H]thymidine (Fig.
3). LPS-mediated NO synthesis was associated with a significant
50% decrease in tritiated thymidine incorporation following a 20-h
incubation in the presence of LPS ( p ⬍ 0.05 vs control and LPS
⫹ L-NAME).
These data suggest that LPS-mediated NO synthesis inhibits total protein synthesis and cell proliferation in this model of ANA-1
murine macrophages. Total protein synthesis initially declines following 12 h of exposure to NO.
LPS-mediated NO production and gene transcription
rRNA expression patterns
The pattern of total RNA expression was then examined in this
experimental model (Fig. 5A). In control cells, the typical electrophoretic pattern of 28S and 18S rRNA expression was found. In
contrast, in the presence of 50 and 100 ng/ml LPS, a distinctive
cleavage pattern of 18S and 28S rRNA was found. This pattern is
associated with decreased levels of 18S and 28S rRNA, as determined by intensity of the bands on gel electrophoresis. Inhibition
of NO production by the addition of L-NAME resulted in restoration of the normal rRNA pattern. The electrophoretic pattern of
genomic DNA expression in LPS- and LPS ⫹ L-NAME-treated
cells did not differ and specifically did not exhibit an apoptotic
pattern (Fig. 5B).
An LPS dose-response experiment was performed using LPS
concentrations varying from 50 ng/ml to 10 ␮g/ml and an incubation period of 20 h (Fig. 6A). Again, a specific cleavage pattern
FIGURE 4. Nuclear run-on analysis in ANA-1 murine macrophages.
Nuclear run-on studies were performed in ANA-1 murine macrophages
exposed to 100 ng/ml LPS and/or 4 mM L-NAME for 20 h. Housekeeping
genes for ␤-actin, GAPDH, and cyclophillin were used to sample global
gene transcription. The blot is representative of five experiments.
of rRNA was exhibited in all cells treated with LPS. In contrast,
the typical rRNA pattern was found in control, L-NAME-, and LPS
⫹ L-NAME-treated cells. A transition in the intensity of RNA
degradation was noted between LPS concentrations of 50 –200 ng/
ml. The experiments were then repeated using LPS concentrations
of 50, 100, 150, 200, and 250 ng/ml to better characterize the
transition (Fig. 6B). No distinct transition corresponding to a specific LPS concentration was evident from these experiments.
When an exogenous source of NO, 400 ␮M SNAP, or 400 ␮M
S-nitroso-N-acetylcysteine was added with LPS ⫹ L-NAME to
ANA-1 macrophages, the distinctive NO-mediated cleavage pattern for rRNA seen in LPS-treated cells was noted (data not
shown). The time dependence of this rRNA cleavage pattern was
examined. Control, LPS-, and LPS ⫹ L-NAME-treated cells were
incubated for a period of 0, 4, 8, 12, 16, and 20 h before RNA
extraction was performed. The RNA cleavage pattern appeared in
LPS-treated cells at 12, 16, and 20 h of incubation. The cells incubated for a period of 20 h exhibited a more intense pattern with
diminished bands corresponding to both 18S and 28S rRNA when
compared with those found in cells treated for only 12 or 16 h. Of
interest is the finding that the time course of rRNA cleavage parallels that found for inhibition of total protein synthesis and maximal NO production (Fig. 2, B and C).
To determine the identity of these various RNA bands, Northern
blot analysis was performed (Fig. 7). A full-length oligonucleotide
probe was constructed that was complementary to murine 18S
rRNA (1869 bp). Due to the size of 28S rRNA, three separate
cDNA probes were made as follows: 28S-1 (nt 0 –1500), 28S-2
(nt 1559 –3268), and 28S-3 (nt 3275– 4712). Based upon the results of the Northern blot analysis, 18S rRNA is cleaved into three
fragments of ⬃1.0, 0.8, and 0.6 kb. In contrast, 28S rRNA is
cleaved into four fragments. One binds 28S-1 (size ⬃3.3 kb); another (size ⬃2.6 kb) binds 28S-2, and a final two are complementary to 28S-3 (sizes ⬃1.5 and ⬃1.0 kb). These data indicate that
NO, from both endogenous and exogenous sources, induces a specific time-dependent cleavage pattern in rRNA resulting in decreased levels of 18S and 28S rRNA expression.
ANA-1 ribosomal profiles
FIGURE 3. ANA-1 cellular proliferation. [3H]Thymidine incorporation
assays were performed in ANA-1 murine macrophages exposed to 100
ng/ml LPS or LPS ⫹ L-NAME for 20 h. Results represent data from three
separate experiments. ⴱ, p ⬍; 0.05 LPS vs LPS ⫹ L-NAME, or control.
ANA-1 macrophages were exposed to 100 ng/ml LPS for 20 h.
Unstimulated control and LPS ⫹ L-NAME-treated cells served as
comparison groups. In cells exposed to LPS, there was a marked
4-fold decrease in 60S ribosomal A280 (0.025 ⫾ 0.012) compared
with that of control (0.102 ⫾ 0.14; p ⬍ 0.05 vs LPS) and LPS ⫹
L-NAME (0.115 ⫾ 0.18; p ⬍ 0.05 vs LPS) cells, indicating a
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To determine the role of NO production in ANA-1 macrophage
gene transcription, nuclear run-on analysis was performed using
␤-actin, GAPDH, and cyclophillin as target “housekeeping” genes
(Fig. 4). Following 20 h of LPS incubation, there was no difference
in gene transcription for any of the target genes tested. Transcription was not significantly different among control, LPS-,
L-NAME-, and LPS ⫹ L-NAME-treated cells. These results indicate that inhibition of total protein synthesis is not the result of
decreased global gene transcription in LPS-treated ANA-1 murine
macrophages.
3982
NO AND RIBOSOMAL RNA DEGRADATION
FIGURE 5. A, RNA gel of ANA-1 murine macrophages. Total RNA was isolated from ANA-1 murine macrophages exposed to 50 and 100 ng/ml LPS and LPS ⫹
L-NAME for 20 h. The bands of the RNA ladder correspond to 0.24, 1.35, 2.37, 4.4, 7.46, and 9.49 kb. Gel is
representative of five experiments. B, DNA gel of ANA-1
murine macrophages. Genomic DNA from ANA-1 murine
macrophages exposed to 100 ng/ml LPS and LPS ⫹ LNAME for 20 h. A 500-bp DNA ladder is presented for
reference. The gel is representative of four experiments.
Ribosome-associated peptidyl transferase activity
Cleavage of 28S and 18S rRNA may alter 60S ribosomal peptidyl
transferase activity and inhibit total protein synthesis. Peptidyl
transferase activity from ANA-1 total ribosomal fractions was determined using LPS concentrations of 0, 1, 5, 10, 25, 50, 100, and
1000 ng/ml at time points varying from 0 to 20 h (Fig. 8A). In
certain instances, 4 mM L-NAME was also added. In unstimulated
control cells, peptidyl transferase activity was maintained throughout the study period. LPS concentrations of 1, 5, 10, and 25 ng/ml
were associated with a progressive, concentration- and time-dependent decrease in peptidyl transferase activity beginning at 12 h
of incubation. In contrast, LPS treatment at concentrations of 50,
100, and 1000 ng/ml was associated with maximally depressed
enzyme activity beginning at 12 h with a subsequent decline thereafter at 16 and 20 h of incubation. At LPS concentrations of 50,
100, and 1000 ng/ml, peptidyl transferase activity was 2-fold less
than that of controls at 20 h of incubation. Ablation of NO synthesis by addition of 4 mM L-NAME restored peptidyl transferase
activity to control levels at all time points studied. The addition of
an exogenous source of NO in the form of SNAP significantly
depressed enzyme activity at all time points compared with both
control and LPS cells. L-NAME alone did not alter peptidyl transferase activity in comparison to those of control cells (data not
shown). To further examine the threshold characteristic of these
data, the IC50 of peptidyl transferase activity was plotted as a function of LPS concentration (Fig. 8B). These data suggest that NO,
FIGURE 6. A, Characterization of LPS dose response of
rRNA cleavage in ANA-1 murine macrophages. Total RNA
was isolated from ANA-1 murine macrophages exposed to 50,
100, 200, 400, 800, 1,200, 2,000, 5,000, and 10,000 ng/ml LPS
and LPS ⫹ L-NAME for 20 h. The bands of the RNA ladder
correspond to 0.24, 1.35, 2.37, 4.4, 7.46, and 9.49 kb. Gel is
representative of three experiments. B, Characterization of LPS
dose response of rRNA cleavage in ANA-1 murine macrophages. Total RNA was isolated from ANA-1 murine macrophages exposed to 50, 100, 150, 200, and 250 ng/ml LPS for
20 h. The bands of the RNA ladder correspond to 0.24, 1.35,
2.37, 4.4, 7.46, and 9.49 kb. The gel is representative of two
experiments.
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decrease in quantity of 60S ribosomes. In addition, predictably, the
A280 of the 80S ribosomal particle composed of 60S and 40S
ribosomes was also decreased in LPS-treated cells (0.012 ⫾ 0.009)
compared with that of control (0.265 ⫾ 0.019; p ⬍ 0.05 vs LPS)
and LPS ⫹ L-NAME-treated (0.281 ⫾ 0.023; p ⬍ 0.05 vs LPS)
cells. These data suggest that LPS-mediated NO synthesis is associated with a decrease in relative quantities of 60S and 80S ribosomal particles, which are requisite for protein synthesis.
The Journal of Immunology
3983
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FIGURE 7. Northern blot analysis of rRNA cleavage pattern in ANA-1
murine macrophages. Northern blot analysis was performed in ANA-1 murine macrophages exposed to 100 ng/ml LPS and/or 4 mM L-NAME for
20 h. Probes were constructed from full-length 18S rRNA, and the following portions of 28S rRNA: 28S-1 (nt 0 –1500), 28S-2 (nt 1559 –3268), and
28S-3 (nt 3275– 4712). The blot is representative of three experiments.
from either exogenous or endogenous sources, inhibits ribosome
fraction-associated peptidyl transferase activity.
Discussion
In this model of LPS-mediated iNOS expression in ANA-1 murine
macrophages, we demonstrate that NO, both from endogenous and
exogenous sources, inhibits total protein synthesis and cell proliferation. This inhibition parallels total NO production beginning
after ⬃12 h of exposure to NO and reaches its maximum following
⬃20 h of exposure. In addition, 28S and 18S rRNA cleavage occur
at all concentrations of LPS examined during a standard 20-h incubation, suggesting that rRNA cleavage is both NO- and timedependent. Transcription, as determined by nuclear run-on analysis of the standard “housekeeping” genes, ␤-actin, GAPDH, and
cyclophillin, is not altered by LPS-mediated NO production.
Northern blot analysis demonstrated the presence of two cleavage
sites in both 18S and 28S rRNA. The time course and NO concentration dependence of decreased total protein synthesis correlates with appearance of this distinctive rRNA cleavage pattern.
We hypothesized that NO-mediated inhibition of cellular protein
synthesis may be the result of fragmentation of the rRNA components essential to 60S ribosome-mediated protein elongation. Subsequent studies found that levels of 60S ribosome and ribosomal
peptidyl transferase activity were significantly decreased in
ANA-1 murine macrophages exposed to endogenous and exogenous sources of NO. We conclude that NO-mediated inhibition of
total protein synthesis is the result of depressed 60S ribosomeassociated peptidyl transferase activity.
Eukaryotic protein synthesis is catalyzed by ribosomes, which
are large complexes of proteins and rRNA (8). The smaller 40S
rRNA subunit binds both mRNA and tRNA, while the larger 60S
rRNA subunit catalyzes peptide bond formation. Together the 60S
and 40S ribosomes compromise the larger functional unit of the
80S ribosome. The process of protein translation centers upon
binding of an aminoacyl-tRNA molecule to the vacant A site on
the 40S ribosomal subunit. The carboxyl end of the polypeptide
chain is uncoupled from the tRNA molecule linked to the growing
end of the polypeptide chain at the adjacent P site; a peptide bond
is formed to the amino acid linked to the tRNA molecule in the A
FIGURE 8. A, Ribosomal peptidyl transferase activity in ANA-1 murine macrophages. Peptidyl transferase activity was determined in the ribosomal fraction from ANA-1 murine macrophages exposed to 1, 5, 10,
25, 50, 100, and 1000 ng/ml LPS, 400 ␮M SNAP, and/or 4 mM L-NAME
for 20 h. Activity is presented as fmol/mg protein/min. The results represent data from three experiments. p ⬍ 0.05; SNAP vs LPS at all concentrations, LPS ⫹ L-NAME, and control. p ⬍ 0.05; 5, 10, 25, 50, 100, and
1000 ng/ml LPS vs LPS ⫹ L-NAME and control. B, IC50 of ribosomal
peptidyl transferase activity in ANA-1 murine macrophages and LPS
concentration.
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mechanism may reside in enzymatic endonuclease or ribozyme
activity. Finally, it will be important to determine the signaling
pathway for the induction of this cleavage pattern.
The functional correlate of NO-mediated inhibition of total protein synthesis in the setting of LPS stimulation is unknown. Is it a
cytotoxic or cytoprotective response? It has been hypothesized that
inhibition of protein synthesis and mitochondrial respiration may
serve as a stress response (5). The purpose may be to transiently
shut-down nonessential cellular functions and conserve cellular
resources in the presence of proinflammatory cytokines and/or endotoxemia. However, when chronically maintained, this process
may lead to ultimate loss of cell viability. In our study, there were
no discernible differences in cell viability based upon trypan blue
exclusion or LDH release.
These considerations notwithstanding, we have demonstrated
that LPS-mediated NO production is associated with decreased
total protein synthesis without change in global gene transcription
programming. This process is paralleled by a specific, reproducible, NO- and time-dependent cleavage pattern in rRNA constituents of the 60S ribosome, 28S and 18S rRNA. This results in
decreased expression of both the 60S ribosome and the complete
80S ribosome, both of which are essential for protein synthesis,
and significant inhibition of 60S ribosome-associated peptidyl
transferase. The time course of decreased peptidyl transferase activity closely parallels that noted with cleavage of 28S and 18S
rRNA. In this model of LPS-treated ANA-1 murine macrophages,
we conclude that one potential mechanism underlying the inhibition of total protein synthesis is NO-mediated degradation of constituent rRNA components of the 60S ribosome and decreased
60S-associated peptidyl transferase activity, an essential central
step in the protein synthetic pathway.
References
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site. This central reaction, termed peptidyl transferase, requires an
intact 28S rRNA in combination with four ribosomal proteins in
the 60S ribosome. Work based upon the prokaryotic model indicates that the 28S rRNA equivalent is a critical component of the
basic catalytic unit (9, 10). The effect of NO on peptidyl transferase activity has not been previously examined. However, a single conserved histidine residue associated with the catalytic site of
peptidyl transferase is sensitive to oxidative stress induced by singlet oxygen generation (8). Although our data suggests that NO
induces a specific pattern of rRNA degradation, the sensitivity of
the active site of peptidyl transferase to oxidative stress suggests
another possible mechanism by which NO may modulate peptidyl
transferase function and ultimately, total protein synthesis.
In our model, we hypothesize that NO mediates a specific pattern of 18S and 28S rRNA cleavage. As a result of depletion of
intact components of the 60S ribosome, peptidyl transferase activity is decreased, total protein synthesis is depressed, and inhibition
of cell proliferation ensues. Alternatively, it is possible that these
RNA bands are the result of altered maturation or cleavage of
rRNA precursors rather than or in addition to that of the mature
rRNA forms. NO-mediated inhibition of protein synthesis has been
previously observed in multiple experimental models using hepatocytes and macrophages. However, the underlying mechanism
has not been not extensively studied. Recently, Kim et al. (5) examined NO-mediated inhibition of total protein synthesis in RAW
264.7 murine macrophages. They found that exposure to NO was
associated with phosphorylation of the protein synthesis initiation
factor, eIF-2␣. 80S ribosome formation was inhibited following a
14-h incubation with LPS. They postulated that NO impairs protein translation by phosphorylation of the ␣ subunit of eIF-2␣ and
inhibits binding of the initiator methionyl-tRNA to 40S rRNA.
These investigators did not specifically address the role of NO in
rRNA processing.
In a similar fashion, our results also indicate diminution in quantities of both 80S and 60S ribosome, which are essential structural
scaffolds upon which translation occurs. In addition, 60S ribosomal peptidyl transferase activity paralleled the time course of
28S and 18S rRNA degradation. We did not specifically address
eIF-2␣ activity. However, NO may certainly act at multiple points
in the protein synthetic pathway, including eIF-2␣ phosphorylation
and inhibition of 60S ribosome formation with consequent ablation
of peptidyl transferase activity.
In this experimental model, NO mediates a specific cleavage
pattern in rRNA that is not paralleled by apoptosis. The effect of
NO on rRNA metabolism has not been previously addressed.
However, work in the early 1980’s by Varesio and colleagues (11–
14) examined murine macrophages activated by individual exposure to IFN-␣, IFN-␥, and LPS. Similar to that found in our study,
they demonstrated a specific cleavage pattern in 28S rRNA with
decreased accumulation of 28S rRNA and associated ribosomal
particles. This pattern was associated with a cytotoxic functional
profile rather than a suppressive profile. Given that this body of
work predates the discovery of NO, the authors did not specifically
address the presence or absence of NO. However, the similarities
with the results from our study are compelling. Varesio et al. (12)
may have been the first to identify a specific rRNA cleavage pattern that is mediated by cytokine- or LPS-mediated NO synthesis
in macrophages. The specific sites of rRNA cleavage in 18S and
28S rRNA are the subject of ongoing investigation in our lab. It is
unclear whether the cleavage site is specific for a defined nucleotide sequence or a rRNA tertiary structural motif. In addition, the
NO AND RIBOSOMAL RNA DEGRADATION